Obesity is driven by excessive intake of fats and carbohydrates and is a major contributor to insulin resistance and type 2 diabetes. However, excess protein consumption is also becoming recognized as a potential contributor to insulin resistance, acting through elevated levels of circulating amino acids (AAs). The goal of this research project is to (i) identify the AA(s) responsible for mediating this response, (ii) to determine the role of increased intracellular Ca2+ ([Ca2+]i) in regulating the AA effects and (iii) to elucidate the underlying molecular mechanism by which [Ca2+]i controls this response through the mammalian target of rapamycin (mTOR) Complex1. It is known that AAs promote insulin resistance through serine/threonine phosphorylation of insulin substrate 1 and 2 (IRSI/2). We and others recently showed that, in cases of excess nutrients, this effect on IRSI phosphorylation is mediated in part through the activation of the mTOR Complex1 and its downstream effector, S6 kinase 1 (S6K1). Importantly, we have also shown that the effects of AAs are mediated through class 3 PI3K, or human Vps34, and not through the canonical class I PI3K signal transduction pathway. Furthermore, we found that hVps34 co-immunoprecipitates with mTOR, suggesting that this complex is critical for AA-induced S6K1 activation. Our recent findings implicate an AA- induced increase in [Ca2+]i, as the triggering event leading to increased mTOR Complex1/signaling via hVps34. Our hypothesis is that AA overload triggers hVps34 activation through a distinct form of mTOR Complex1 leading to downstream hyperactivation of S6K1 and insulin resistance. Our research plan in testing this hypothesis in L6 myotubes is to (i) identify, analyze, and localize the protein partners that make up the hVps34-mTOR Complex1 using immunoprecipitation, mass spectrometry, cloning, siRNA ablation, immunofluorescence and FRET; (ii) determine which AA(s) is responsible for eliciting the rise in [Ca2+]i, and identify the source of the [Ca2+]i and (iii) elucidate the mechanism by which AAs and Ca2+ affect the activity of the hVps34-mTOR signaling complex. The major health risks associated with nutrient overload are type 2 diabetes and the onset of cardiovascular disease. Moreover, recent epidemiological studies show that obesity is a major risk factor for cancer. These pathologies have been driven by the worldwide expansion of the obesity epidemic, fueled by a dramatic rise in caloric intake and the increased availability and low cost of food. As this proposal is designed to identify the specific molecules responsible for excess nutrient- induced insulin resistance, it will provide the potential for the development of novel pharmacological therapies. Furthermore, current treatments are largely focused on ameliorating insulin resistance by increasing insulin production or activity. In contrast, the studies described here could contribute to new treatment strategies that directly target insulin resistance in peripheral tissues. PUBLIC HEALTH RELEVANCE: Insulin resistance and type 2 diabetes are becoming exceedingly insidious health problems because of the worldwide obesity epidemic. This research will increase our understanding of the cellular mechanisms that link overeating, specifically excess protein consumption, to insulin resistance. This work could contribute to the development of new and more effective drugs to treat insulin resistance and type 2 diabetes.